Treatment of severe pain is a significant clinical challenge, and use of traditional analgesics is limited by side effects. One alternative strategy targets cannabinoid receptors (CBRs) indirectly by inhibiting the enzyme monoacylglycerol lipase (MAGL), which degrades the endogenous cannabinoid 2-arachidonoyl glycerol (2-AG). We propose that MAGL inhibitors have the advantage of producing anatomically and temporally precise increases in 2-AG release and CBR activation targeted to neural circuits activated by pain. More specifically, this renewal application proposes to investigate expression and mechanisms of analgesic effects produced by acute and chronic delivery of MAGL inhibitors in novel preclinical procedures developed and validated during the initial funding period to assess pain-depressed behaviors that model clinically relevant manifestations of pain. The overarching hypothesis is that MAGL inhibition will safely and effectively ease clinically relevant signs of pain-related behavioral depression via 2-AG-mediated activation of CB1Rs that buffer activity in CNS pain circuits. Aim 1 will compare the effects of MAGL inhibitors, direct CBR agonists, and a systematic series of control drugs in complementary assays of pain-stimulated and pain-depressed behavior. We hypothesize that MAGL inhibitors will produce analgesia in all assays with minimal side effects, and preliminary data support this hypothesis. Aim 2 will test the hypothesis that MAGL inhibitors alleviate pain-related depression of behavior by CBR-mediated alleviation of pain-related depression of mesolimbic dopamine circuits. We propose to localize markers of neuronal activity and measure eCB levels in candidate regions for modulating pain-depressed behavior. Additionally, we propose to examine effectiveness of MAGL inhibitors to relieve pain- related depression of mesolimbic dopamine release assessed by in vivo microdialysis, and of brain site-targeted MAGL inhibitor injections to alleviate pain-depressed behavior. Aim 3 will assess the effects of repeatedly administered MAGL inhibitors on both behavior and CB1R regulation in a novel model of chronic episodic pain. We hypothesize that MAGL inhibition will produce sustained relief of pain-depressed behavior without significant CB1R desensitization and/or downregulation in brain areas that mediate relief of pain-depressed behavior. Successful completion of the proposed studies will inform further consideration of MAGL inhibitors as viable options for pain treatment.